The present invention provides bactericidal/permeability-increasing protein (BPI) dimer products characterized by enhanced in vivo biological activity and stable pharmaceutical compositions containing the same.
Lipopolysaccharide (LPS) is a major component of the outer membrane of gram-negative bacteria and consists of serotype-specific O-side-chain polysaccharides linked to a conserved region of core oligosaccharide and lipid A. Raetz. Ann. Rev. Biochem., 59:129-170 (1990). LPS is an important mediator in the pathogenesis of gram-negative septic shock, one of the major causes of death in intensive-care units in the United States. Morrison, et al., Ann. Rev. Med. 38:417-432 (1987).
LPS-binding proteins have been identified in various mammalian tissues Morrison, Microb. Pathol., 7:389-398 (1989); Roeder, et al., Infect., Immun., 57:1054-1058 (1989). Among the most extensively studied of the LPS-binding proteins is bactericidal/permeability-increasing protein (BPI), a basic protein found in the azurophilic granules of polymorphonuclear leukocytes. Human BPI protein has been isolated from polymorphonuclear neutrophils by acid extraction combined with either ion exchange chromatography [Elsbach, J. Biol. Chem., 254:11000 (1979)] or E. coli affinity chromatography [Weiss, et al., Blood, 69:652 (1987)] and has potent bactericidal activity against a broad spectrum of gram-negative bacteria.
The amino acid sequence of the entire human BPI protein, as well as the DNA encoding the protein, have been elucidated in FIG. 1 of Gray, et al., J. Biol. Chem., 264:9505 (1989), incorporated herein by reference (SEQ ID NOS: 1 and 2). The Gray et al. publication discloses the isolation of human BPI-encoding cDNA from a cDNA library derived from DMSO-induced cells of the human promyelocytic leukemia HL-60 cell line (ATTC CCL 240). Multiple PCR amplifications of DNA from CDNA library derived from such DMSO-induced HL-60 cells as well as DNA from normal human blood and bone marrow cell have revealed the existence of human BPI-encoding cDNAs wherein the codon specifying valine at amino acid position 151 is either GTC (as set out in SEQ ID No: 1) or GTG. Moreover, cDNA species employing GTG to specify valine at position 151 have also been found to specify either lysine (AAG) for the position 185 amino acid (as in SEQ ID Nos: 1 and 2) or a glutamic acid residue (GAG) at that position.
A proteolytic fragment corresponding to the N-terminal portion of human BPI holoprotein possesses the antibacterial activity of the naturally-derived 55 kDa human BPI holoprotein. In contrast to the N-terminal portion, the C-terminal region of the isolated human BPI protein displays only slightly detectable anti-bacterial activity. Ooi, et al., J. Exp. Med., 174:649 (1991). A BPI N-terminal fragment designated rBPI.sub.23, Gazzano-Santoro et al., Infect. Immun. 60:4754-4761 (1992), and comprising approximately the first 199 amino acids of the human BPI holoprotein, has been produced by recombinant means as a 23 kD protein.
The bactericidal effect of BPI has been shown to be highly specific for sensitive gram-negative species. The precise mechanism by which BPI kills bacteria is not yet completely elucidated, but it is known that BPI must first attach to the surface of susceptible gram-negative bacteria. This initial binding of BPI to the bacteria involves electrostatic and hydrophobic interactions between the basic BPI protein and negatively charged sites on LPS. LPS has been referred to as "endotoxin" because of the potent inflammatory response that it stimulates, i.e., the release of mediators by host inflammatory cells which may ultimately result in irreversible endotoxic shock. BPI binds to lipid A, the most toxic and most biologically active component of LPS.
In susceptible bacteria. BPI binding is thought to disrupt LPS structure, leading to activation of bacterial enzymes that degrade phospholipids and peptidoglycans, altering the permeability of the cell's outer membrane, and initiating events that ultimately lead to cell death. [Osbach and Weiss, Inflammation: Basic Principles and Clinical Correlates, eds. Gallin et al., Chapter 30, Raven Press, Ltd. (1992)]. BPI is thought to act in two stages. The first is a sublethal stage that is characterized by immediate growth arrest, permeabilization of the outer membrane and selective activation of bacterial enzymes that hydrolyze phospholipids and peptidoglycan. Bacteria at this stage can be rescued by growth in serum albumin supplemented media but not by growth in whole blood, plasma or serum. The second stage, defined by growth inhibition that cannot be reversed by serum albumin, occurs after prolonged exposure of the bacteria to BPI and is characterized by extensive physiologic and structural changes, including penetration of the cytoplasmic membrane.
BPI-induced permeabilization of the bacterial cell envelope to hydrophobic probes such as actinomycin D is rapid and depends upon the initial binding of BPI to LPS, leading to organizational changes which probably result from binding to the anionic groups in the KDO region of LPS, normally responsible for stabilizing the outer membrane through binding of Mg.sup.++ and Ca.sup.++. Binding of BPI and subsequent bacterial killing depends, at least in part, upon the LPS polysaccharide chain length, with long 0 chain bearing organisms being more resistant to BPI bactericidal effects than short, "rough" organisms (Weiss et al., J. Clin. Invest. 65: 619-628 (1980). This first stage of BPI action is reversible upon dissociation of the BPI, a process requiring synthesis of new LPS and the presence of divalent cations (Weiss et al., J. Immunol. 132: 3109-3115 (1984). Loss of bactericidal viability, however, is not reversed by processes which restore the membrane integrity, suggesting that the bactericidal action is mediated by additional lesions induced in the target organism and which may be situated at the cytoplasmic membrane (Mannion et al., J. Clin. Invest. 86: 631-641 (1990)). Specific investigation of this possibility has shown that, on a molar basis, BPI is at least as inhibitory of cytoplasmic membrane vesicle function as polymyxin B (In't Veld et al., Infection and Immunity 56:1203-1208 (1988)) but the exact mechanism has not yet been elucidated.
BPI is also capable of neutralizing the endotoxic properties of LPS to which it binds. Because of its bactericidal properties for gram-negative organisms and its ability to neutralize LPS, BPI can be utilized for the treatment of mammals suffering from diseases caused by gram-negative bacteria, such as bacteremia or sepsis.
U.S. Pat. No. 5,198,541 (PCT/US88/02700) the disclosures of which are hereby incorporated by reference, describe recombinant genes encoding and methods for expression of BPI proteins, including BPI holoprotein and fragments of BPI. They also describe the use of N-terminal fragments of BPI protein for co-treatment with certain antibiotics, specifically penicillin. cephalosporins, rifampicin and actinomycin D.
Gram-negative bacteria include bacteria from the following species: Acidaminococcus, Acinetobacter, Aeromonas, Alcaligenes, Bacteroides, Borderella, Branhamella, Brucella, Calymmawobacterium, Campylobacier, Cardiobactenium, Chromobacterium, Citrobacter, Edwardsiella, Enterobacter, Escherichia, Flavobacterium, Francisella, Fusobacierium, Haemophilus, Klebsiella, Legionella, Moraxella, Morganella, Neisseria, Pasturella, Plesiomonas, Proteus, Providencia, Pseudomonas, Salmonella, Serratia, Shigella, Streptobacillus, Veillonella, Vibrio, and Yersinia species.
Antibiotics are natural chemical substances of relatively low molecular weight produced by various species of microorganisms, such as bacteria (including Bacillus species), actinomycetes (including Streptomyces) and fungi, that inhibit growth of or destroy other microorganisms. Substances of similar structure and mode of action may be synthesized chemically, or natural compounds may be modified to produce semi-synthetic antibiotics. These biosynthetic and semi-synthetic derivatives are also effective as antibiotics. The major classes of antibiotics are (1) the .beta.-lactams, including the penicillins, cephalosporins and monobactams; (2) the aminoglycosides, e.g., gentamicin. tobramycin, netilmycin, and amikacin; (3) the tetracyclines; (4) the sulfonamides and trimethoprim; (5) the fluoroquinolones, e.g., ciprofloxacin, norfloxacin, and ofloxacin; (6) vancomycin; (7) the macrolides, which include for example, erythromycin, azitbromycin, and clarithromycin; and (8) other antibiotics, e.g., the polymyxins, chloramphenicol and the lincosamides.
Antibiotics accomplish their anti-bacterial effect through several mechanisms of action which can be generally grouped as follows: (1) agents acting on the bacterial cell wall such as bacitracin, the cephalosporins, cycloserine, fosfomycin, the penicillins, ristocetin, and vancomycin; (2) agents affecting the cell membrane or exerting a detergent effect, such as colistin, novobiocin and polymyxins; (3) agents affecting cellular mechanisms of replication, information transfer, and protein synthesis by their effects on ribosomes, e.g., the aminoglycosides, the tetracyclines, chloramphenicol, clindamycin, cycloheximide, fucidin, lincomycin, puromycin, rifampicin, other streptomycins, and the macrolide antibiotics such as erythromycin and oleandomycin; (4) agents affecting nucleic acid metabolism, e.g., the fluoroquinolones, actinomycin, ethambutol, 5-fluorocytosine, griseofulvin, rifamycins; and (5) drugs affecting intermediary metabolism, such as the sulfonamides, trimethoprim, and the tuberculostatic agents isoniazid and para-aminosalicylic acid. Some agents may have more than one primary mechanism of action, especially at high concentrations. In addition, secondary changes in the structure or metabolism of the bacterial cell often occur after the primary effect of the antimicrobial drug.
Heparin is a heterogenous group of straight-chain anionic mucopolysaccharides (glycosaminoglycans) having anticoagulant properties. Although others may be present, the main sugars occurring in heparin are: (1) .alpha.-L-iduronic acid 2-sulfate, (2) .sup.2 -deoxy-2-sulfamino-.alpha.-D-glucose 6-sulfate, (3) .beta.-D-glucuronic acid, (4) .sup.2 -acetamido-2-deoxy-.alpha.-D-glucose, and (5) .alpha.-L-iduronic acid. These sugars are present in decreasing amounts, usually in the order (2)&gt;(1)&gt;(4)&gt;(3)&gt;(5), and are joined by glycosidic linkages, forming polymers of varying sizes. Heparin is strongly acidic because of its content of covalently linked sulfate and carboxylic acid groups. Heparin is found within mast cell granules and is released upon degranulation. A cell associated form of heparin is termed heparan sulfate. Heparan sulfate is a broad term used to describe a variety of sulfated proteoglycans (HSPG's) found with a near-ubiquitous distribution on mammalian cell surface membranes and in the extracellular matrix. HSPG contains a variable percentage of pentamaric heparin-like sequences that function in a similar fashion as soluble heparin. The HSPG's serve as a repository for antithrombin III (ATIII) and for heparin-binding growth factors such as fibroblast growth factors (FGF) 1-S, IL-8, GM-CSF and IL-3. Folkman et al., Inflammation: Basic Principles and Clinical Correlates, 2d Ed. Chapter 40, pp 821-839 (1992). In fact, cells made genetically deficient in HSPG's require exogenous heparin for growth.
Heparin is commonly administered in doses of up to 400 U/kg during surgical procedures such as cardiopulmonary bypass, cardiac catherization and hemodialysis procedures in order to prevent blood coagulation during such procedures. The anticoagulant effect of heparin in blood is a result of the interaction of heparin with ATIII. The heparin/ATIII complex is a potent inhibitor of many of the clotting factors of the coagulation cascade. Specific inhibition has been demonstrated for activated Factors IXa, Xa, XIa, XIIIa and thrombin. The heparin/ATIII complex has the highest affinity for Factor Xa and thrombin which are common to both the intrinsic and extrinsic clotting pathways involved as the last two steps of the clotting cascade that results in the conversion of fibrinogen to fibrin. The additional antibacterial and antiendotoxin effects of BPI would be particularly advantageous in post-surgical heparin neutralization. When heparin is administered for anticoagulant effects during surgery, it is an important aspect of post-surgical therapy that the effects of heparin are promptly neutralized so that normal coagulation function can be restored.
Angiogenesis is closely associated with endothelial cell proliferation and constitutes the development of new capillary blood vessels. As such, it is an important process in mammalian development and growth, and in menstruation processes. The release of angiogenic growth factors, such as fibroblast growth factors 1-5, induces proliferation of endothelial cells via a heparin-dependent receptor binding mechanism. See Yayon et al., Cell, 64:841-848 (1991). These heparin-binding growth factors can be released due to vascular trauma (wound healing), immune stimuli (autoimmune disease), inflammatory mediators (prostaglandins) and from tumor cells.
Angiogenesis is also associated with a number of pathological conditions in which it would be desirable to inhibit such new blood vessel development. As one example, angiogenesis is critical to the growth, proliferation, and metastasis of various tumors. Other pathological conditions associated with angiogenesis include diabetic retinopathy, retrolental fibroplasia, neovascular glaucoma, psoriasis, angiofibromas, immune and non-immune inflammation including rheumatoid arthritis, capillary proliferation within atherosclerotic plaques, hemangiomas, endometriosis and Kaposi's Sarcoma.
Chronic inflammation is usually accompanied by angiogenesis. Arthritis is a chronic syndrome characterized by the inflammation of the peripheral joints accompanied by synovial thickening and the influx of immune factors and cells such as polymolphonuclear leukocytes (PMN). In rheumatoid arthritis, the inflammation is immune driven, while in reactive arthritis, inflammation is associated with infection of the synovial tissue with pyrogenic bacteria or other infectious agents. Folkman et al., Inflammation: Basic Principles and Clinical Correlates, 2d Ed. Chapter 40, pp 821-839 (1992) note that many types of arthritis progress from a stage dominated by an inflammatory infiltrate in the joint to a later stage in which a neovascular pannus invades the joint and begins to destroy cartilage. While it is unclear whether angiogenesis in arthritis is a causative component of the disease, and not an epiphenomenon, there is evidence that angiogenesis is necessary for the maintenance of synovitis in rheumatoid arthritis.
Co-owned U.S. Pat. No. 5,348,942, continuation-in-part U.S. patent application Ser. No. 08/093,202 filed Jul. 15, 1993 (PCT/US94/02401), continuation-in-part U.S. patent application Ser. No. 08/183,222 filed Jan. 14, 1994 and continuation-in-part U.S. patent application Ser. No. 08/209,762 filed Mar. 11, 1994 (PCT/US94/02465) and U.S. patent application Ser. No. 08/306,473 filed Sep. 15, 1994 (PCT/US94/10427), the disclosures of which are hereby incorporated by reference, address use of BPI protein products for treatment of conditions associated with heparin binding and/or neutralization, including neutralization of the anti-coagulant properties of heparin, inhibition of angiogenesis, tumor and endothelial cell proliferation and treatment of chronic inflammatory disease states such as arthritis.
Various other utilities have been described for therapeutic administration of BPI protein products. Co-owned, copending U.S. patent application Ser. No. 08/031,145 filed Mar. 12, 1993 (PCT/US94/02463), and continuation-in-part U.S. patent application Ser. No. 08/285,803 filed Aug. 4, 1994, the disclosures of which are hereby incorporated by reference, address use of BPI protein products in treatment of mycobacterial diseases. Co-owned, copending U.S. patent application Ser. No. 08/125,651, filed Sep. 22, 1993, continuation-in-part U.S. patent application Ser. No. 08/273,401 filed Jul. 11, 1994, and continuation-in-part U.S. patent application Ser. No. 08/311,611 filed Sep. 22, 1994 (PCT/US94/11225) address combinations of BPI protein products and antibiotics. Co-owned, copending U.S. patent application Ser. No. 08/273,540 filed Jul. 11, 1994 and continuation-in-part U.S. patent application Ser. No. 08/372,105 filed Jan. 13, 1995 (PCT/US95/00498), the disclosures of which are hereby incorporated by reference, address use of BPI protein products in treatment of fungal infections. Co-owned, copending U.S. patent application Ser. No. 08/274,299 filed Jul. 11, 1994 and continuation-in-part U.S. patent application Ser. No. 08/372,783 filed Jan. 13, 1995 (PCT/US95/00656), the disclosures of which are hereby incorporated by reference, address use of BPI protein products in treatment of gram-positive infections. Co-owned, copending U.S. patent application Ser. No. 08/132,510, filed Oct. 5, 1993, and continuation-in-part U.S. patent application Ser. No. 08/318,357 filed Oct. 5, 1994 (PCT/US94/11404) address use of BPI protein products in the treatment of conditions involving depressed reticuloendothelial system function. Co-owned, copending U.S. patent application Ser. No. 08/232,527 filed Apr. 22, 1994 addresses use of BPI protein products for treating conditions associated with intestinal ischemia and reperfusion. Co-owned and copending U.S. patent application Ser. No. 08/093,201 filed Jul. 14, 1993, continuation-in-part U.S. patent application Ser. No. 08/274,303 filed Jul. 13, 1994 (PCT/US94/07834) address methods for potentiating BPI protein product bactericidal activity by administration of LBP protein products. Co-owned, copending U.S. patent application Ser. No. 08/188,221 filed Jan. 24, 1994, continuation-in-part U.S. patent application Ser. No. 08/291,112 filed Aug. 16, 1994, and continuation-in-part U.S. patent application Ser. No. 08/378,228 filed Jan. 24, 1995 (PCT/US95/01151) incorporated by reference herein, address use of BPI protein products in the treatment of humans exposed to gram-negative bacterial endotoxin in circulation. The disclosures of all of the patents and patent applications referenced herein are specifically incorporated by reference herein.
Efforts to produce pharmaceutical grade BPI products for human treatment have not yielded uniformly satisfactory results. A principal reason for this is the nature of the amino acid sequence of human BPI and the nature of the recombinant host cell environment in which the products are produced. As one example, biologically-active rBPI products produced as secretory products of CHO host cells transfected with a construct encoding the initial 199 residues of BPI [RBPI (1-199)] may be purified in good yields. As noted in co-owned, copending U.S. patent application Ser. No. 08/072,063, filed May 19, 1993 (PCT/US93/04752) elution of BPI products from S-Sepharose beads incorporated into roller bottles containing transformed CHO cells yielded substantially monomeric BPI products when a 1.0 M NaCl-Acetate buffer was employed, but yielded multimeric protein forms when a 1.5 M NaCl-Acetate buffer was then employed. Moreover, secreted expression products resulting from CHO cells transfected with DNA encoding a secretory leader sequence and BPI amino acid residues 1-199 actually yielded mixtures of carboxy terminal-shortened BPI protein products terminating at residue 193 or at other residues intermediate between residue 193 and 199. Co-owned, copending U.S. patent application Ser. No. 08/013,801, filed Feb. 2, 1993 (PCT/US94/01235), addresses analog BPI protein products and DNA sequences encoding the same wherein cysteines at positions 132 or 135 are replaced by different amino acids for the purpose of reducing multimer and cysteine adduct formation in recombinant products and also addresses development of recombinant expression products using DNAs encoding the initial amino terminal residue (1) to from about 175 to 193 of BPI, which products display reduced carboxy terminal heterogeneity.
There continues to be a need in the art for improved BPI protein product preparations. Such products would be obtainable in large yield as recombinant products from transformed host cells, would retain the bactericidal, endotoxin binding, endotoxin neutralizing, heparin binding, heparin neutralizing and other biological activities of BPI rendering them suitable for therapeutic use, and would ideally display enhanced in vivo biological activity, thus providing for improved therapeutic methods involving administration of BPI protein products, alone or as a cotreatment with other agents, including, for example, antibiotics.